Abstract We designed a highly sensitive reverse transcription nested polymerase chain reaction targeting the M-segment (NPCR-M) of severe fever with thrombocytopenia syndrome (SFTS) virus. NPCR-M was performed in parallel with three other referenced PCR assays QPCR-S, PCR-M, and NPCR-S to assess their clinical usefulness as routine diagnostic techniques for SFTS. In this multi-centered prospective study, 122 blood samples from 38 laboratory-confirmed SFTS patients and 85 control samples were used. The results demonstrated that QPCR-S and NPCR-S had better sensitivity rate up to 21 days after symptom onset however, the PCR-M showed poor sensitivity after 7 days of symptom onset. Our designed NPCR-M had a higher detection rate up to 40 days from symptom onset and revealed the persistence of SFTSV RNA in the early convalescent phase. No false-positive results were seen for the control samples. Additionally, NPCR-M showed positive results for a sample that initially showed negative results from other PCRs and for many other samples collected in the convalescent phase of SFTS. Our designed nested PCR is suitable for SFTSV detection in patients’ blood collected in the acute and early convalescent phase of SFTS, and shows better sensitivity and high specificity even up to 40 days after symptom onset.